Radiometric assays for glycerol, glucose, and glycogen

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.     

Flow injection chemiluminescent assays for glycerol and triglycerides using a co‐immobilized enzyme reactor

A flow injection method for the determination of glycerol using a co‐immobilized enzyme reactor containing glycerokinase and glycerol‐3‐phosphate oxidase is described. The hydrogen peroxide produced is monitored by using a luminol chemiluminescence reaction in the presence of catalyst such as Co(II). The detection limit (2.5 × blank noise) for glycerol is 7 × 10^?3 mmol/L with a sample throughput of 40/h. The calibration graph is linear over the range studied (0.2–1.0 mmol/L) with relative standard deviation 1.2–2.4%. The method is applied to the determination of glycerol in blood serum produced off‐line from triglycerides using lipase isolated from bovine pancreas. Copyright © 2003 John Wiley & Sons, Ltd.     

Glucose determination in samples taken by microdialysis by peroxidase-catalyzed luminol chemiluminescence

An automatic, luminometric assay of glucose in samples of the extracellular water space obtained by microdialysis is described. The assay involves oxidation by glucose oxidase (EC 1.1.3.4) and mutarotation of glucose by aldose mutarotase (EC 5.1.3.3.). The H2O2 formed is subsequently determined in a reaction catalyzed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay is linear between 0.01 and 1 nmol in the cuvette. The detection limit, defined as 3 standard deviations of the reagent blank, was 0.008 mumol/liter in the cuvette. A complete oxidation of glucose is obtained within 4 min and 25 samples are automatically assayed within 75 min. Addition of microdialysate sample obtained from human adipose tissue in vivo did not interfere with the standard curves. Glucose added to microdialysate resulted in a complete recovery compared to a H2O2 standard. Analytical interference from different factors was investigated. No interference was observed up to the following concentrations: 5 mumol/liter epinephrine, 1 mumol/liter norepinephrine, 100 mumol/liter insulin, 500 mumol/liter pyruvate, 50 mmol/liter lactate, and 1 mumol/liter ascorbate. The glucose values with the present method correlated strongly (r = 0.984) with values obtained using a routine method involving glucose oxidase and peroxidase.     

Fluorometric procedures for measuring triglyceride concentrations in small amounts of tissue and plasma

It has been previously shown that triglycerides can be specifically hydrolyzed by lipase from Rhizopus arrhizus in the presence of hog liver esterase and sodium dodecyl sulfate. The glycerol produced can then be measured by sequential reactions with glycerokinase, pyruvate kinase, and lactate dehydrogenase: glycerol and ATP are converted to glycerol-3-phosphate and ADP by glycerokinase; the ADP reacts with phosphoenolpyruvate and pyruvate kinase to yield pyruvate; the pyruvate is converted to lactate with lactate dehydrogenase, and the cofactor NAD+ is simultaneously reduced to NADH. This report describes procedures by which either the disappearance of NADH or the appearance of NAD+ was determined fluorometrically, with 10- to 100-fold greater sensitivity than by spectrophotometry. In addition, enzymatic cycling of NAD+ was used to increase the sensitivity of the assay over 1000-fold, and thereby provided accurate measurement of less than 1 ng of triglyceride. Results obtained from the three fluorometric methods were highly correlated with an automated periodate oxidation method using serum samples and lipid extracts of muscle tissue.     

The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.     

Characteristics of a glycerol utilization mutant of Neurospora crassa.

A mutant of Neurospora crassa able to grow on liquid minimal glycerol medium without evidence of conidiation and with high cell yields has been isolated and shown to be allelic to ff-1. The glycerol-specific induction of glycerokinase and glycerol-3-phosphate dehydrogenase was similar in both wild-type and mutant cells, although higher specific activities as well as higher glycerokinase cross-reacting material levels were found in fully induced mutant cells. After growth in minimal glycerol medium there is a significant reduction in wild-type cells of the activities of both pyruvate dehydrogenase and dihydrolipoyl transacetylase. This evidence indicates a relationship between the conditional acetate requirement by wild-type cells grown on glycerol medium and the levels of the pyruvate dehydrogenase complex.     

Flow-injection enzymatic analysis for glycerol and triacylglycerol

A flow-injection enzymatic analytical system was developed for determination of glycerol and triacylglycerol based on enzymatic reactions in capillary followed by electrochemical detection. The hydrogen peroxide produced from the enzyme reaction was monitored by a platinum-based electrochemical probe. Different immobilization strategies on silica support were studied. The best and most effective configuration found for the measurement of glycerol and triacylglycerols in this system was the tandem connection of a lipase column and a silica-fused capillary column coimmobilized with glycerokinase (GK) and glycerol-3-phosphate oxidase (GPO). Lipase helps the breakdown of triacylglycerol to yield free fatty acids and glycerol, while glycerokinase catalyzes the adenosine-5-triphosphate-dependent phosphorylation of glycerol to yield alpha-glycerol phosphate, which can subsequently be oxidized by 3-glycerol phosphate oxidase to produce hydrogen peroxide. Response-surface methodology (RSM) was applied to optimize the proposed system for glycerol. Experiment settings were designed by central composite design to investigate the combined effects of pH, flow rate, reaction temperature, and ATP concentration on collected signals. The fitted model, per RSM, showed that the optimum conditions of the system are 2 mM ATP in 0.1 M carbonate buffer (pH 11.0), flow rate of 0.18 mL/min, temperature of 35 degrees C, 20 microL of sample injection, and applied voltage of 0.650 V. The proposed biosensing system using lipase, GK, and GPO exhibited a flow-injection analysis peak response of 2.5 min and a detection limit of 5 x 10(-5) M glycerol (S/N = 3) with acceptable reproducibility (CV < 4.30%). It also had linear working ranges from 10(-4) to 10(-2) M for glycerol and from 10(-3) to 10(-2) M for triacylglycerol. The capillary enzyme reactor was stable up to 2 months in continuous operation, and it was possible to analyze up to 15 samples per hour. The present biosensing system holds promise for on-line detection of triacylglycerol in serum and glycerol content in fermented products.     

Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction.

The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.     

Reliable Drosophila body fat quantification by a coupled colorimetric assay

Factors and mechanisms controlling lipometabolism homeostasis share a remarkable evolutionary conservation between humans and Drosophila flies. Accordingly, the Drosophila model has been successfully used to understand the pathophysiology of human metabolic diseases such as obesity. Body fat stores in species as different as humans and flies consist of neutral lipids, mainly triacylglycerols. Changes in body fat storage are a diagnostic phenotype of lipometabolism imbalances of genetic or environmental origin. Various methods have been developed to quantify Drosophila body fat storage. The most widely used method adopts a commercial coupled colorimetric assay designed for human serum triacylglycerol quantification, which is based on glycerol content determination after enzymatic conversion of glycerides into glycerol. The coupled colorimetric assay is compatible with large-scale genetic screen approaches and has been successfully applied to characterize central regulators of Drosophila lipometabolism. Recently, the applicability of the coupled colorimetric assay for Drosophila storage fat quantification has been questioned in principle. Here we compare the performance of the coupled colorimetric assay on Drosophila samples with thin layer chromatography, the "gold standard" in storage lipid analysis. Our data show that the presented variant of the coupled colorimetric assay reliably discriminates between lean and fat flies and allows robust, quick and cost-effective quantification of Drosophila body fat stores.     

Characterization of the multiple-chamber perifused fat cell system.

A multiple-chamber perifused fat cell system is described. Six chambers containing fat cells were perifused in parallel with buffer. Perifusate was collected for assay of glycerol as an index of lipolytic rates and cells in each chamber can be taken for analysis of biochemical intermediates. The system is so designed that drugs can be infused into the buffer and equally distributed in each chamber or can be individually infused into the buffer to one chamber, allowing for six different conditions to be tested in the same population of fat cells. The time and distribution characteristics of infused material are described. Time relationships are described for isoproterenol and glycerol release and for cyclic AMP levels in the fat cells, and the dose-response relationship between isoproterenol and glycerol release is shown.     

Genetic and enzymatic characterization of the inducible glycerol dissimilatory system of Neurospora crassa.

The glycerol dissimilatory system in Neurospora crassa was analyzed through the characterization of 18 Glp- mutants which were isolated after inositol-less death and filtration enrichment. All mutants obtained by this procedure could be assigned to one of three complementation groups. The subsequent genetic characterization of these glp mutations revealed lesions on the I, II, and VI chromosomes at the glp-1, glp-2, and glp-4 loci, each of which was subjected to fine-structure analysis. Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively. Additional evidence, obtained from studies of the inducibility of glycerokinase by glycerol, cold treatment, or deoxyribose, suggests that glp-1 is involved in controlling the expression of glp-4.     

Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro.

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.     

Quantitation of lyso-platelet activating factor molecular species from human neutrophils by mass spectrometry

Two physicochemical methods have been developed for the quantitative analysis of lyso-platelet activating factor (lyso-PAF) based on gas-liquid chromatography-mass spectrometry (GLC/MS) and fast atom bombardment-mass spectrometry (FAB/MS) using stable isotope dilution. After addition of deuterated internal standards, lyso-PAF produced from neutrophils was purified by silicic acid chromatography and thin-layer chromatography (TLC). The GLC/MS assay employed phospholipase C or hydrofluoric acid for hydrolysis of the phosphocholine moiety to yield ether monoglycerides. Condensation of monoglycerides with acetone yielded the 1-O-alkyl-2,3-isopropylidene glycerol which could be analyzed by GLC/MS. The ions corresponding to M-15 fragments for both the labeled and unlabeled derivatives were monitored in a selected ion recording mode. Standard curves were found to be linear over the range tested (10-2000 ng) with a limit of detection found to be below 200 pg injected on column. For the FAB/MS assay, the unmodified lyso-PAF was well suited for direct analysis; however, the limit of detection (S/N greater than 3) using a glycerol matrix was found to be 5 ng placed on the probe tip. It was found that human neutrophils contain approximately 300 pg/10(6) cells which increased 2-3-fold during the 5-min period following challenge with 1.9 microM calcium ionophore, A23187. Two molecular species of lyso-PAF were identified as hexadecyl and octadecyl ethers at sn-1 with the octadecyl molecular species of lyso-PAF predominating in abundance after stimulation.     

Determination of L-carnitine by flow injection analysis with NADH fluorescence detection

A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 microM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 microM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU/microM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1.47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations.     

Resveratrol directly affects in vitro lipolysis and glucose transport in human fat cells

Resveratrol is a naturally occurring polyphenol found in many dietary sources and red wine. Recognized as a cancer chemoprevention agent, an anti-inflammatory factor and an antioxidant molecule, resveratrol has been proposed as a potential anti-obesity compound and to be beneficial in diabetes. Most of the studies demonstrating the anti-adipogenic action of resveratrol were performed as long-term treatments on cultured preadipocytes. The aim of this study was to analyse the acute effects of resveratrol on glucose uptake and lipolysis in human mature adipocytes. Samples of subcutaneous abdominal adipose tissue were obtained from overweight humans and immediately digested by liberase. Fat cells were incubated (from 45 min to 4 h) with resveratrol 1 μM–1 mM. Then, glycerol release or hexose uptake was determined. Regarding lipolysis, the significant effects of resveratrol were found at 100 μM, consisting in a facilitation of isoprenaline stimulation and an impairment of insulin antilipolytic action. At 1 and 10 μM, resveratrol only tended to limit glucose uptake. Resveratrol 100 μM did not change basal glucose uptake but impaired its activation by insulin or by benzylamine. This inhibition was not found with other antioxidants. Such impairment of glucose uptake activation in fat cells may led to a reduced availability of glycerol phosphate and then to a decreased triacylglycerol assembly. Therefore, resveratrol increased triacylglycerol breakdown triggered by β-adrenergic activation and impaired lipogenesis. Consequently, our data indicate that resveratrol can be considered as limiting fat accumulation in human fat cells and further support its use for the mitigation of obesity.     

A new linear plot for standard curves in kinetic substrate assays extended above the Michaelis-Menten constant: application to a luminometric assay of glycerol

In conventional kinetic substrate assays the standard curve is plotted as observed reaction rate, upsilon obs, versus added substrate concentration, Sadd, and has a linearity limited to Sadd much less than Km. From this plot the blank reaction rate, upsilon bl, is easily estimated but not the contaminating substrate concentration, Scon, present in reagents (unless it is the only blank source). Thus the actual substrate concentration, S = Scon + Sadd, cannot be estimated as required for the various linear plots based on the Michaelis-Menten equation. We have derived an expression, (upsilon obs - upsilon bl)/Vapp = Sadd/(Kmapp + Sadd), containing only those parameters measured for a conventional standard curve (Vapp and Kmapp are obtained from a plot of (upsilon obs - upsilon bl) versus (upsilon obs - upsilon bl)/Sadd). A plot of (upsilon obs - upsilon bl)/Vapp versus Sadd/(Kmapp + Sadd) can be used as a standard curve with the following advantages over the conventional standard curve: (a) For all kinetic substrate assays it is identical and connects the points (0, 0) and (1, 1). Thus deviations from true Michaelis-Menten kinetics or erroneous kinetic constants are easily detected. (b) Since it is linear even above Km, the analytically useful range is considerably extended. (c) For assays with a wide dynamic range it can be used in lin-lin or log-log form. The procedure is illustrated for a kinetic assay of glycerol (Kmapp = 40 mumol/liter). The plot was found to be entirely linear in the range 0.07-100 mumol/liter (glycerol concentration in cuvette).     

Isocratic ion-exchange chromatographic assay for the nucleotide gemcitabine triphosphate in human white blood cells

An isocratic bio-analytical assay for the nucleotide gemcitabine triphosphate (2',2'-difluorodeoxycytidine 5'-triphosphate, dFdCTP) in human white blood cells (leukocytes) has been developed and validated. The method is based on ion-exchange liquid chromatography and ultraviolet detection (275 nm). dFdCTP is isolated from the matrix by extraction with perchloric acid while the sample is chilled on ice. After neutralization with potassium hydroxide and removal of the potassium perchlorate precipitate, with the sample still chilled on ice, the mixture is injected into the chromatograph. The method has been validated in the range 0.4-20 microM, 0.4 microM (approximately 20 pmol/10(6) cells) being the lower limit of quantification, using erythrocytes as a substitute for leukocytes. Precisions and accuracies both meet the current requirements for a bioanalytical assay. The stability of dFdCTP in intact mononuclear blood cells on ice is strongly limited (half-life approximately 100 min) and after freezing the half-life of the analyte in the cellular lysate is approximately 30 min. On the other hand, no degradation was observed for dFdCTP for at least approximately 24 h in perchloric acid extracts on ice or in neutralized extracts at ambient temperature. The applicability of the assay was demonstrated in white blood cells of a patient with advanced non-small cell lung cancer receiving i.v. gemcitabine.     

Early development of adipose cell lipogenesis and glycerol utilization in Zucker obese rats.

A study of adipose cell metabolism was made at ages 5, 7, 10, and 14 wk of age in genetically obese Zucker rats. Adipose samples were surgically removed and used for in vitro adipose cell incubations and for characterization of enzyme patterns. Lipogenic capacity from glucose and enzymes normally associated with lipogenesis (malic enzyme, citrate cleavage enzyme and glucose-6-PO4 dehydrogenase) followed the same pattern of development. At 5 wk of age, the adipose cells of obese animals had a greater capacity for fat synthesis than the lean rats. At all other ages lipogenic activity and enzyme levels were either similar or less than the pair-fed lean littermates. Glycerol utilization by isolated fat cells was similar; however, adipose tissue glycerokinase was elevated in obese rats at 14 wk of age. It was concluded that there was no apparent change in specific lipogenic capacity of fat cells from the obese rat when compared to its lean littermate. It was also concluded that increased adipose glycerokinase activity in obese rats represented a secondary shift in metabolism.     

Adrenocorticotropin. 57. Synthesis and biological activity of ostrich and turkey hormones

Synthesis of ostrich and turkey corticotropin (ACTH) has been accomplished by the solid-phase method. Each was identical to the natural hormone in high performance liquid chromatography. Relative potencies in a lipolytic assay in isolated rabbit fat cells were: human ACTH, 100; ostrich ACTH, 53; turkey ACTH, 28. In isolated rat fat cells relative lipolytic potencies were: human ACTH, 100; ostrich ACTH, 2; turkey ACTH, 13. It was concluded that lipolytic potency is sensitive to alterations in structure throughout the entire length of the ACTH sequence in the rat fat cell assay.     

Characterization of beta-adrenergic receptors by (3H) isoproterenol in adipocytes of humans and rats

The specific binding of (3H) isoproterenol to isolated fat cells of human and rat was characterized. Binding of (3H) isoproterenol to isolated fat cells of rat was saturable with 420 pmol of (3H) isoproterenol bound/100 mg of lipid. Half-maximal saturation occurred at 5 microM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its binding sites. Kinetic analysis of (3H) isoproterenol binding provided a value of 2.01 x 10(4) min-1. M-1 for the forward bimolecular rate constant, k1. Dissociation of (3H) isoproterenol was a first order reaction with a rate constant, k2, of 0.62 x 10(-1) min-1. The ratio k2/k1 = 3.07 microM provides an independent measurement of the KD for the interaction of (3H) isoproterenol with its binding sites which is in agreement with the values obtained by steady state analysis (3 to 5 microM). The apparent equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its receptor in human fat cells obtained by steady state analysis was 1 to 0.9 microM. Scatchard- and Hill-analysis suggest the possibility of different negatively cooperative interactions among the binding sites in human and rat. beta-Adrenergic agonists competed for the binding sites. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Compounds such as DOPA, dopamine and (m-Hydroxyphenyl)2-methyl-aminoethanol which are structurally related to catecholamines had little or no affinity for (3H) isoproterenol binding sites.